Β-ionone loaded l-arginine peptide dendrimers nanoparticle system and the method of obtaining this system

Abstract

The invention relates to a nanoparticle system synthesised for use in active targeting, consisting of 1st generation G1 peptide dendrimer with the chemical structure of Formula (I), 2nd generation G2 peptide dendrimer with the chemical structure of Formula (II), 3rd generation G3 peptide dendrimer with the chemical structure of Formula (III), 4th generation G4 peptide dendrimer with the chemical structure of Formula (IV), the core tof which consists of ethylene diamine, and branching units and surface functional groups of which consist only of L-arginine, and the synthesis method of this system. It relates to the nanoparticle system obtained as a result of the complex formation of any of the 3rd generation G3 peptide dendrimers from these dendimers, modified with folic acid with the chemical structure of Formula (V) by modification of the 3rd generation G3 dendrimer with the targeting molecule folic acid with β-ionone (4-(2,6,6-Trimethyl-1- cyclohexenyl)-3-buten-2-one) and the method of obtaining this system. They show anticancer properties by means of the nano formulation structure they create. Said nanoparticle has a cytotoxic effect against cancer cells, especially cervical cancer HeLa cancer cells.

Description

[0001] p-IONONE LOADED L -ARGININE PEPTIDE DENDRIMERS NANOPARTICLE SYSTEM AND THE METHOD OF OBTAINING THIS SYSTEM

[0002] Technical Field

[0003] The invention relates to a peptide dendrimer the core of which consists of ethylene diamine (EDA), branching units and surface functional groups only from L-Arginine, and the synthesis method of this peptide dendrimer and the nanoparticle system it forms with [3-ionone. Said peptide dendrimer can be used as a drug or gene carrier by means of its nanoparticle properties.

[0004] State of the Art

[0005] In pharmaceutical technology, directing an active substance at the correct concentration to the cell or tissue in the desired region of the living organism at the desired time interval is a subject that has been intensively studied for a long time. This is achieved with drug carrier systems with suitable properties. There are four important considerations in the use of drug delivery systems in a biological system. These are drug resistance due to physiological barriers, drug resistance at the cellular level, distribution of the drug in the body and possible binding state, and removal of the drug from the body

[0001] . In order to overcome these difficulties, an ideal drug carrier system should have a biocompatible structure and be non-toxic, should have the ability to form a stable nano-sized carrier and scaffold with high active ingredient capacity, should have appropriate cellular adhesion of the therapeutic carrier in the cytoplasm or nucleus, endocytosis and intercellular movement, acceptable biodegradation, controlled and differential drug release properties, its ability to bind to non-specific cellular and blood proteins should be minimal, and its synthesis should be stable and reproducible for clinical use [2]. For this purpose, polymeric and lipophilic carrier systems are used. These prepared carrier systems improve the bioavailability of water- soluble or insoluble active substances in pharmaceutical research by increasing their solubility and provide many advantages such as increasing its therapeutic effectiveness and cell penetration by prolonging its residence time in the blood. The properties and versatility of polymers make them ideal delivery systems that can provide drug release in various ways, especially in a controlled manner, and as gene carriers. Polymeric carriers can be natural or synthetic based. Natural polymers are difficult to adapt to versatile applications, but they can be used for some purposes with modification with suitable synthetic compounds. However, synthetic polymers can be designed for specific purposes and can be evaluated for a wide range of uses. Dendrimers constitute an important class among synthetic polymers.

[0006] Dendrimers are called macromolecules with a branched structure. Dendrimer structures have an extremely symmetrical and ordered structure. Generally, many different dendrimer molecules are synthesised by selecting the monomers in the branching units of dendrimers from structures with different properties. A wide range of monomers with different properties can be used to create the molecular structure of dendrimers, but they may exhibit cytotoxic properties due to their high surface charges. Although this situation creates a restrictive element for monomer selection, different properties can be provided to the structure with various surface modifications [2], There are many dendrimer structures such as PAMAM dendrimer, peptide dendrimer, polyester dendrimer, poly(ethylene imine) (PEI) dendrimers, polypropylene imine) (PPI) dendrimer.

[0007] Peptide dendrimers are a type of dendrimer structure and are synthesised by polymerisation of amino acids or small peptide units. Peptide dendrimers have properties such as good biocompatibility and water solubility due to their flexible structure, rich end groups for the binding of various chemo / biomolecules, and similarity to protein structures. These properties make peptide dendrimers an important position in targeted delivery systems, diagnostic tools and vaccine studies. Peptide dendrimers are molecules that theoretically have an amino acid core, branching units, surface functional groups, or any combination of the three, and are defined as three distinct types depending on the position of amino acids in their structure. The first of these is graft peptide dendrimers, which are traditional dendrimers that have non-natural amino acids or organic groups as branching core molecules in their structure and whose surface functional groups are modified with peptides or proteins. In the second type of peptide dendrimers, the core and branching units consist of natural amino acids. The third type of peptide dendrimers are molecules with a multi-amino acid core and peptide branching units [5], These structures give peptide dendrimers an important position in the pharmaceutical field [6],

[0008] Active targeting is defined as the ability of the drug substance to selectively and quantitatively accumulate in the target tissue or organ, regardless of its chemical structure and the way it is transported into the cell. Active substances with active targeting properties are of great importance for both drug and gene release in modern therapy, as they minimise or eliminate the negative side effects that classical chemotherapy agents may cause in healthy cells. Nanoparticle carrier systems are among the most effective structures with active targeting properties. This active targeting is achieved by binding a targeting compound / agent that can be directed to the nanoparticle carrier system used, an antigen or receptors in the target tissue, in other words, by functionalising the drug carrier in this way [7], In active targeting, since the entry of the drug into the cell (internalisation) and its release occur through the formation of an endosome (endocytosis), the definition of receptor-mediated targeting and release mechanism by endocytosis is also used for this targeting. For example, cancer cells that tend to proliferate rapidly produce certain receptors in large numbers in order to efficiently use nutritional resources such as sugar, vitamins and folic acid and folate conjugate nanoparticle systems whose surface is modified with folic acid not only target tumour cells easily but also are frequently and successfully used for receptor-mediated targeting since endosomes are formed.

[0009] In a study carried out in the state of the art, anti-angio-genetic heparin-binding arginine dendrimers are synthesised. These dendrimers are obtained by solid phase peptide synthesis of two dendrimers containing L-glycine as the core, L-lysine as branching units, and different numbers (8 or 16) of L-arginine as surface groups. It is concluded that the peptide dendrimer with 16 arginine groups on its surface has a more tightly packed surface structure, thus a higher heparin binding capacity and shows a more active activity in preventing angiogenesis, than the dendrimer with 8 arginine groups. However, it is not known how effective this structure is when used in nano carrier systems.

[0010] In another study in the state of the art, poly-L-lysine dendrigrafts were synthesised as nanoscale drug carriers for cancer treatment, and cytotoxicity and cell penetration were evaluated in vitro studies. The data obtained showed that, despite their higher molecular weight, poly-L-lysine dendrigrafts showed a cytotoxic effect close to the cytotoxicity values specified in the sources for other dendrimers and increased cell internalisation in experiments conducted on a colon cancer cell line. However, there is no information on whether arginine can be used in the branching units of said dendrigrafts.

[0011] Due to reasons such as the limitations and inadequacies of the solutions in the state of the art, the lack of studies on the application of nano carrier systems with arginine on their surfaces, and the fact that to what extent the presence of the arginine structure on the surfaces of existing nano carrier systems can be effective in active targeting is unknown, it has become necessary to make a development in this field.

[0012] Brief Description and Aims of the Invention

[0013] In the invention, a peptide dendrimer synthesised for use in active targeting and the synthesis method of this peptide dendrimer are described. The core of said peptide dendrimer consists of ethylene diamine, branching units and surface functional groups consist only of L-Arginine, and by means of its nanoparticle properties, it can be used as a nanoparticle carrier for drug / gene delivery.

[0014] The main aim of the invention is to provide a nanoparticle system that can be used in cancer treatment. The nanoparticle system described in the invention has a peptide dendrimer structure, and by means of the nanocomplexes it forms with the anticancer agent [3-ionone (Bl), said nanoparticle system is used in cancer treatment. Thus, the nanoparticle system that is the subject of the invention can be used both as a vehicle system and in treatment methods that require many active targeting, especially in cancer treatment.

[0015] Another aim of the invention is to provide a nanoparticle system that will enable active targeting of the active drug in drug treatments. The nanoparticle carrier system of the invention is synthesised as a peptide dendrimer whose core consists of ethylene diamine, branching units and surface functional groups only L-Arginine. By means of the nano dimensions of this peptide dendrimer, its branching units and its surface functional groups consisting only of L-Arginine, it can be used as a nanoparticle carrier for active targeting with various modifications in drug / gene delivery.Another aim of the invention is to provide a nanoparticle system that can be used in the treatment of cervical cancer. The nanoparticle system of the invention has a cytotoxic effect on HeLa cancer cells, which are cervical cancer cells. By means of this cytotoxic effect, the nanoparticle system of the invention can be used in the treatment of cervical cancer.

[0016] Description of Drawings

[0017] Figure 1: FTIR spectra of G1 peptide dendrimer

[0018] Figure 2: FTIR spectra of G2 peptide dendrimer

[0019] Figure 3: FTIR spectra of G3 peptide dendrimer

[0020] Figure 4: FTIR spectra of G4 peptide dendrimer

[0021] Figure 5: FTIR spectra of FA-G3 peptide dendrimer

[0022] Figure 6: FTIR spectra of Boc-L-Arg(Boc)-OH structure

[0023] Figure 7: FTIR spectra of G1* and EDA structures

[0024] Figure 8: FTIR spectra of Guanidine HCI, Di-tertbutyl-dicarbamate and L-arginine structures

[0025] Detailed Description of the Invention

[0026] The invention relates to a nanoparticle system, the core of which consists of ethylene diamine (EDA), branching units and surface functional groups of which only of L- arginine, and which comprises any one of

[0027] - 1stgeneration G1 peptide dendrimer with the chemical structure of

[0028] Formula I, or

[0029] Formula I

[0030] - 2ndgeneration G2 peptide dendrimer with the chemical structure of

[0031] Formula II, or

[0032] Formula II

[0033] - 3thgeneration G3 peptide dendrimer with the chemical structure of

[0034] Formula III, or

[0035] Formula III

[0036] - 4thgeneration G4 peptide dendrimer with the chemical structure of

[0037] Formula IV, or

[0038]

[0039] - 3rdgeneration G3 peptide dendrimer modified with folic acid (FA) with the chemical structure of Formula V

[0040] - and [3-ionone (4-(2,6,6-Trimethyl-1-cyclohexenyl)-3-buten-2-one). Said nanoparticle system has a cytotoxic effect against cancer cells, especially HeLa cells, which are cervical cancer cells, and has active targeting properties. The synthesis method of 1stgeneration G1 peptide dendrimer with the chemical structure of Formula I, 2ndgeneration G2 peptide dendrimer with the chemical structure of Formula II, 3rdgeneration G3 peptide dendrimer with the chemical structure of Formula III, 4thgeneration G4 peptide dendrimer with the chemical structure of Formula IV, which are the subject of the invention, comprises the process steps of: i. Protecting the amine groups of the L-Arginine molecule with N-Boc (diterbutyl-dicarbamate), ii. Synthesising the 1stGeneration G1 * dendrimer with the chemical structure of Formula VI,

[0041] Formula VI iii. Obtaining the 1stgeneration G1 with the chemical structure of Formula I by removing the N-Boc protection in the 1stgeneration G1 * dendrimer,

[0042] Formula I iv. Synthesising the 2stGeneration G2* dendrimer with the chemical structure of Formula VII,

[0043]

[0044] Formula VII v. Obtaining the 2ndgeneration G2 with the chemical structure of Formula II by removing the N-Boc protection in the 2ndgeneration G2* dendrimer,

[0045] Formula II vi. Synthesising the 3rdGeneration G3* dendrimer with the chemical structure of Formula VIII,

[0046]

[0047] Formula VIII vii. Obtaining the 3rdgeneration G3 with the chemical structure of Formula III by removing the N-Boc protection in the 3rdgeneration G3* dendrimer,

[0048] Formula III viii. Synthesising the 4thGeneration G4* dendrimer with the chemical structure of Formula IX, io

[0049]

[0050] Formula IX ix. Obtaining the 4thgeneration G4 with the chemical structure of Formula IV by removing the N-Boc protection in the 4thgeneration G4* dendrimer, x. Obtaining the G3-FA complex with the chemical structure of Formula V by modification of the 3rdgeneration G3 dendrimer with FA (folic acid),

[0051]

[0052] Formula V xi. Formation of nanoconjugate structure by drug loading into dendrimers.

[0053] During the synthesis of the nanocamer system of the invention, first, the peptide dendrimers are synthesised. The dendrimer, the core molecule EDA (ethylene diamine) and the branching units of which comprises L-Arginine, of said peptide dendrimer is synthesised according to the activated polyamidation mechanism in a solvent environment by following a synthesis step extending from the nucleus to the branching units according to the divergent method. In the first step of the synthesis, the -NH2 groups in the L-Arginine molecule are protected with the Boc compound, in other words, N-Boc'ing process is carried out and the product [Boc-L-Arg(Boc)-OH] is obtained. This product is used in the synthesis of every G* product. To obtain each G end product, the protective groups in the molecule are removed to release the -NH2 groups. All monomer and activator addition steps are carried out under nitrogen atmosphere. Products with and without preservative groups are shown as G* and G, respectively, with the relevant G number (Formula I, II, III, IV, VI, VII, VIII and IX). The processing steps performed in the synthesis steps are presented in detail below.

[0054] The N-Boc process, which is carried out in order to protect the amine groups in the L- Arginine dendrimer, is carried out in a short time under the catalysis of Gu.HCI in ethanol. In order to increase the yield, the solvent removal, washing and lyophilisation steps in the purification processes were optimised, and the [Boc-L-Arg(Boc)-OH] molecular structure of said product was confirmed by FTIR spectra (Figure 1 -5).

[0055] The method of carrying out said N-Boc reaction comprises the process steps of:

[0056] Adding 1 mmol L-Arg to a 2 mL ethanol solution containing 2.5 mmol Boc and (0.15 mol%) Gu.HCI while stirring,

[0057] Leaving it to stir in a water bath at 35-40°C and monitoring, and completing the N-Boc'ing process when the colour turns transparent,

[0058] Removing ethanol from the mixture by stirring at 250 rpm for 5 hours 30 minutes at room temperature, and

[0059] Obtaining [Boc-L-Arg(Boc)-OH] as a pure product by washing with 2 mL deionized water and 2 mL hexane, lyophilizing and storing it at +4°C.

[0060] In the synthesis, products G1 -G4 peptide dendrimers are obtained according to the activated polyamidation reaction using the N-Boc'ing process product Boc-L-Arg(Boc)- OH dissolved in DMF.

[0061] Synthesis method of G1 * dendrimer comprises the process steps of:

[0062] Dissolving the compound [Boc-L-Arg(Boc)-OH] (2 mmol) in 10 mL DMF in an ice bath (0°C),

[0063] Adding 2 mmol HBTU (Hexafluorophosphate Benzotriazole Tetramethyl Uranium) and 2 mmol HOBt (Hydroxybenzotriazole) as amidation reaction activators to the medium and mixing for 5 minutes,

[0064] Then adding DIPEA (N,N-diisoproylethylamine) (4 mmol) and mixing for 15 minutes,

[0065] Adding EDA (1 mmol) and mixing in an ice bath for 30 minutes,

[0066] Continuing the reaction for 2 days while slowly bringing the reaction mixture to room temperature,

[0067] Cooling the mixture in an ice bath and purifying the product by precipitating it with 0.5 M citric acid, and

[0068] Separating the precipitated product by centrifugation at 7500 rpm for 5 min periods, washing with acetonitrile and lyophilizing by dispersing in deionized water. As a result of the application of the mentioned process steps, 1stgeneration G1 * peptide dendrimer is obtained in the form of white powder. To remove the protective groups in the 1stgeneration G1 * dendrimer, in other words, to obtain the 1stgeneration G1 , the removal of the protective groups - which protect the NH2 groups - on the 1stgeneration G1 * synthesis product is done by dissolving in TFA (Trifluoroacetic acid).

[0069] The method of removing the protective groups that protect the -NH2 groups on the 1stgeneration G1 * synthesis product comprises the process steps of;

[0070] Dissolving 1 mmol of the 1stgeneration G1 * product in 40 mmol of TFA (Boc groups: TFA in a 1 : 10 molar ratio) and mixing in an ice bath in N2 atmosphere for 2 hours, and then removing the solvent and washing the precipitate with cold diethyl ether 3 times and lyophilising it.

[0071] Synthesis method of the 2ndgeneration G2* dendrimer comprises the process steps of:

[0072] Dissolving the 1stgeneration G1 peptide dendrimer (1 mmol) in 10 mL DMF in an ice bath (0°C),

[0073] Then, adding DIPEA (4 mmol) to the medium and allowing to mix for 10 minutes,

[0074] Then, adding HBTLI (4 mmol) and HOBt (4 mmol) as amidation reaction activators and mixing for 10 minutes,

[0075] Adding [Boc-L-Arg(Boc)-OH] (4 mmol) to this mixture and stirring in an ice bath for 30 minutes,

[0076] Continuing the reaction for 2 days while slowly bringing the reaction mixture to room temperature, and cooling the mixture in an ice bath.

[0077] Said 2ndgeneration G2* peptide dendrimer is purified by applying the purification process applied to the 1stgeneration G1 * product, and the 2ndgeneration G2* dendrimer is obtained as white powder. The protective groups on the 2ndgeneration G2* dendrimer are removed as described in the 1stgeneration G1 * dendrimer, and the 2ndgeneration G2 dendrimer is obtained. Synthesis method of the 3rdgeneration G3* dendrimer comprises the process steps of:

[0078] Dissolving the 2ndgeneration G2 peptide dendrimer (1 mmol) in 10 mL DMF in an ice bath (0°C),

[0079] Adding DIPEA (8 mmol) to the medium and allowing to mix for 10 minutes, Then, adding HBTLI (8 mmol) and HOBt (8 mmol) as amidation reaction activators and mixing for 10 minutes,

[0080] Then, adding [Boc-L-Arg(Boc)-OH] (8 mmol) and stirring in an ice bath for 30 minutes,

[0081] Continuing the reaction for 2 days while slowly bringing the reaction mixture to room temperature.

[0082] The 3rdgeneration G3* dendrimer, obtained in white powder form, is purified by applying the purification process applied to the 2ndgeneration G2* product. The protective groups on the 3rdgeneration G3* dendrimer are removed as described in the 1stgeneration G1 * dendrimer, and the 3rdgeneration G3 dendrimer is obtained.

[0083] Synthesis method of the 4thgeneration G4* dendrimer comprises the process steps of:

[0084] Dissolving the 3rdgeneration G3 peptide dendrimer (1 mmol) in 10 mL DMF in an ice bath (0°C),

[0085] Adding DIPEA (16 mmol) to the medium and allowing to mix for 10 minutes, Then, adding HBTLI (16 mmol) and HOBt (16 mmol) as amidation reaction activators and mixing for 10 minutes,

[0086] Then, adding [Boc-L-Arg(Boc)-OH] (16 mmol) and stirring in an ice bath for 30 minutes,

[0087] Continuing the reaction for 2 days while slowly bringing the reaction mixture to room temperature.

[0088] The 4thgeneration G4* dendrimer, obtained in white powder form, is purified by applying the purification process applied to the 1stgeneration G1 * product. The protective groups on the G4* dendrimer are removed as described in the 1stgeneration G1 * dendrimer, and the 4thgeneration G4 dendrimer is obtained.

[0089] Modification of the 3rdgeneration G3 dendrimer with FA is carried out by DCC (dicyclohexylcarbodiimide) activation. For this, first, folic acid is dissolved in DMSO, and DCC is added and stirred for 1 hour under nitrogen atmosphere to activate the carboxyl groups in the FA molecule. Then, the 3rdgeneration G3 dendrimer is added to the medium and the reaction continues while stirring for 24 hours at room temperature. At the end of the period, the final product (FA-G3) is purified, lyophilised and stored at +4°C.

[0090] A nanocomplex structure (nG:BI) is prepared between 2ndgeneration G2 and 3rdgeneration G3 peptide dendrimers and [3-ionone (Bl) by self-clustering by the interaction of the two molecules.

[0091] The method of preparation of nanoconjugate structure by drug loading into synthesised 2ndgeneration G2 and 3rdgeneration G3 dendrimers comprises the process steps of;

[0092] Adding nonionic surfactant (Tween-80) with a G:Tween-80 ratio of 1 :1.2 (w / w) to the mixer and mixing for 1 hour while the product dissolved in water was mixed at 400 rpm in a magnetic stirrer so that the concentration of 2ndgeneration G2 and 3rdgeneration G3 peptide dendrimers in the final particle solution was 1 mg / mL,

[0093] After mixing, adding the solution dissolved in 200 pL ethanol and containing Bl at a ratio of 1 :0.9 or 1 :1 or 1 :1.1 or 1 :1.2 or 1 :1.3 or 1 :1.5 to G:BI by mass dropwise, and

[0094] Measuring nanoparticle sizes after mixing for 1 hour or 2 hours or 3 hours.

[0095] An embodiment of the method of preparation of nanoconjugate structure by drug loading into synthesised 2ndgeneration G2 and 3rdgeneration G3 dendrimers comprises the process steps of;

[0096] Adding nonionic surfactant (Tween-80) with a G:Tween-80 ratio of 1 :1.2 (w / w) to the mixer and mixing for 1 hour while the product dissolved in water was mixed at 400 rpm in a magnetic stirrer so that the concentration of 2ndgeneration G2 and 3rdgeneration G3 peptide dendrimers in the final particle solution was 1 mg / mL,

[0097] After mixing, adding the solution dissolved in 200 pL ethanol and containing Bl at a ratio of 1 :1 to G: Bl by mass dropwise, and Measuring nanoparticle sizes after mixing for 2 hours. With 2 hours of mixing, the optimal particle size value for the nG2:BI nanoformulation structure with a 1 :1 (w / w) (G:BI) ratio is obtained as 193.25±4.5 nm, and as 216.4±2.93 nm for nG3: B I. In vitro studies of these nanostructure formulations are carried out using the HeLa cell line to evaluate the cytotoxic effects on it with the MMT test. The results are evaluated based on the data at the 24thand 48thhours. While the synthesis products, 2ndgeneration G2 and 3rdgeneration G3 dendrimers, do not have a negative effect on the viability of HeLa cells, that is, they do not show a significant cytotoxic effect, it is observed that that Bl-loaded conjugates nG2:BI and nG3:BI are much more effective than Bl in terms of cytotoxicity to cell viability. At the 24thhour, the ICso value, which is the expression of the concentration of the substance that reduces the number of viable cells by 50%, for nG2:BI is calculated as 78.37±5.8 pg / mL, for nG3:BI as 52.17±5.88 pg / mL, and for Bl as 341 ,4±5.9 8 pg / mL. At the 48thhour, the ICso value is calculated as 66.19±8.1 8 pg / mL for nG2:BI, as 49.60±5.4 8 pg / mL for nG3:BI, and as 119.3±9.9 8 pg / mL for Bl. In order to examine the change of this activity when a targeting agent is used, the 3rdgeneration G3 dendrimer and FA as the targeting agent are selected, taking into account the particle size and PDI values, as well as the MTT test results, along with the ICso results. In this context, nFA-G3:BI nanoconjugate is prepared with Bl. Likewise, its cytotoxic effects on HeLa cells are examined by MMT test. The ICso value obtained at the end of 24 hours is calculated as 50.4±8.258 pg / mL, and at the 48thhour it is calculated as 41.22±9.5 8 pg / mL. When the results are evaluated comparatively, it is seen that although the cytotoxic effect of Bl on HeLa cells was quite low under operating conditions, the cytotoxic effect of the nG2:BI and nG3:BI nanoconjugates formed with the 2ndgeneration G2 and 3rdgeneration G3 dendrimers synthesised in the invention is much higher than the state of the art, and this effect increases with time. In case of using FA targeting agent, it is seen that for the selected nFA-G3:BI nanoconjugate, this effect is very close to the non-targeted conjugate (ICso =52.17±5.8) at the end of 24 hours (ICso =50.4±8.2 8 pg / mL), and that, at the end of 48 hours, it increased even more (ICso = 41.22±9.5 8 pg / mL), showing that its effectiveness was close to but slightly higher than the conjugate without targeting agent (49.60±5.4 8 pg / mL). These results are thought to be due to the presence of L-arg units in the end groups of the dendritic chains of the synthesised dendrimer molecule, which can be an alternative to the most known targeting agent FA and has high cell affinity. In the invention, selected generation products of new peptide dendrimers containing the synthesised core molecule EDA and branching units L-arg can be used as nanocarriers that can be targeted to HeLa cells without the need for the FA compound, which is a commonly used targeting agent to deliver drugs to cancer cells, under in vitro study conditions. Considering the success of the new nanoformulations nG2:BI and nG3:BI and nFA-G3:BI in carrying Bl, an anticancer agent, it is obvious that the invention is of great importance both for its applicability as targetable nanocamers in nanobiotechnology and for researchers working in this field.

[0098] The 1stgeneration G1 * dendrimer is evaluated in the FTIR spectra by examining the change / disappearance / appearance of the absorption bands mentioned below formed by the 1stgeneration G1 * structure. It was seen in the EDA spectra that NH2 asymmetric stretching band in 3364 cm-1was disappeared, instead in the 3435-3395 cm-1region at maximum 3399 cm-1, a new broad shoulder that belongs to Amid II NH stretching vibrations that can only be seen in secondary amide structure that was appeared at maximum 3379 cm-1(structural vibrational band of L-Arg was observed at 3282 cm-1in its spectra) in 1stGeneration G1 * spectra shifted as expected and observed, at maximum 3265 cm-1with a similar intensity.

[0099] Other bands in the ~3000-2850 cm-1region of the Boc-L-Arg(Boc)-OH molecule spectra are also seen slightly shifted at lower intensities due to the size of the molecule. In the EDA spectra of the stretchings of N-CH bonds at 2820 cm-1, from the bands in the region of 1650-1550 cm-1, the band at maximum ~ 1650 / 1648 cm'1and 1576 cm-1that was from scissoring of NH bonds, the band at the 1158 cm-1that was from rocking of NH bonds / wagging of NH2 bonds, the bands at 823 and 708 cm'1that was from wagging of NH2 groups were disappeared as well and among them the ones that were belong to the structural vibrations of NH bonds overlapped with the others with less intensity. The small sharp band at 1813 cm-1(at 1808 cm-1in Boc FTIR) belonging to the C=O bonds in the other alkyl carbonate structure, which separates from the dicarbonate structure, is also located in the upper wide part of the band that is maximum at 1771 cm'1. The new absorption band at 1722 cm'1is due to the vibrations of the special structure of the secondary amide in the form of R-NH-CO-O-R formed in Boc-L-Arg(Boc)-OH. The C=O bonds in the free COOH group in L-Arg at 1689 cm'1disappeared because the band resulting from the vibrations reacted as expected. The band at 1643 cm'1related to the new secondary Amide I C=O stretching in Boc-L- Arg(Boc)-OH and dendrimer structure is interfering in the wide upper part of the band at 1722 cm-1. The bands at 1556 and 1510 cm-1appeared as a band related to the deformation of NH bonds in the secondary Amide II in the new structure. The intensity of the band has increased significantly since it stems from the wagging of the CH2 groups at 1397 cm-1and the number of CH2 groups in the molecule is high. The band at 1253 cm-1due to C-OH stretching has disappeared. The band at 1223 cm-1is due to Amide III CN stretching (only in secondary amides, it is usually a total absorption of two bonds i.e.-CN and -NH bonds). Other structural vibrations of the compounds used in the synthesis and seen in their FTIR spectra are generally seen in the same form in the product G1 * spectra. As a result, it is confirmed by the 1 st generation G1 * FTIR spectra that the reactions between Boc-L-Arg-Boc, formed by protection with Boc, and the core molecule EDA occur in such a way as expected that a secondary amide structure is formed between the COOH in L-Arg and the NH2 groups in the EDA molecule, without any degradation in the structure.

[0100] When the FTIR spectra of the 1stgeneration G1 dendrimer was evaluated by comparing them with the FTIR spectra of the 1stgeneration G1 * dendrimer, in the ~3560-3000 cm-1 region in the FTIR spectra of the 1stgeneration G1 dendrimer, the intensity of absorption increases due to the asymmetric / sym metric stretching vibrations of the -N-H- bonds in the primary amine -NH2 groups and the imine structure (C=NH) and is seen as a broad long band at maximum 3280 cm-1. Similarly, it is seen that the intensity of absorption in the 3100-2300 cm-1region increases mainly due to 6- guanidino imine structural vibrations and comprises many small absorption bands of various intensities, the most prominent of which are maximum at 3098, 3049, 3004 and 2945 cm-1. It is seen that the intensity of the absorption band resulting from the stretching vibrations of -C=O bonds in the COOH groups in the L-Arg molecule, seen at 1684 cm-1in the L-Arg FTIR spectra, clearly decreased in the FTIR spectra of the 1stgeneration G1 dendrimer due to the reaction. In the same spectrum, a new absorption band appeared at maximum 1673 cm-1, resulting from the bending vibrations of -NH2 groups. Secondary amide bands indicating the formation of new peptide bonds in the FTIR spectra of the 1stgeneration G1 dendrimer are also more prominent, with small bands in the 1685-1650 cm-1region and maximum at 1564, 1551 and 1526 cm-1, and at 1287, 1271 and 1223 cm-1, and are found interfering with others in this region (mainly NH2 and 6-guanidino groups). The new sharp absorption bands at maximum 1183, 1160 and 913 cm-1in the FTIR spectra of the 1stgeneration G1 dendrimer are mainly due to asymmetric bending / stretching of C-NH2 bonds, stretching of -C-N- bonds, and asymmetric bending vibrations of C-N-H bonds. However, It is clearly seen that, in the FTIR spectra of the 1 st generation G1 * dendrimer, the band belonging to the stretching vibrations of -CH bonds in the -CH3 groups in the tertiary butyl C-(CH3)3 structure in the Boc compound at maximum 2993 cm-1and the band resulting from the stretching vibrations of the C=O bonds in the other alkyl carbonate structure, which separates from the small sharp dicarbonate structure at 1813 cm-1disappear in the FTIR spectra of the 1stgeneration G1 dendrimer, as expected. This indicates that the Boc protecting groups have disappeared.

[0101] As a result, the protective groups on the amine groups of the 1stgeneration G1 * dendrimer were successfully removed with the help of TFA and the structure of the 1stgeneration G1 dendrimer is confirmed by FTIR spectra.

[0102] When the FTIR spectra of G2*, G2, G3*, G3, G4* and G4 dendrimers are compared with the FTIR spectra of the characteristic 1stgeneration G1 * and 1stgeneration G1 dendrimers, they exhibit similar characteristic absorption bands due to the similarity in structure. Considering the increase in transmittance values in FTIR spectra as the G number of the synthesised dendrimer molecules increases, it is expected that the intensity of the vibrations in the growing molecule will increase and give results at higher values. There are some acceptable differences in the dendrimer spectra at different Gs due to reasons such as band peaks shifting, low intensity ones interfering with others and therefore not being visible.

[0103] The structure characterisation of the conjugate of 3rdgeneration G3 dendrimers with FA is evaluated by comparing the FTIR spectra of the conjugate with the FTIR spectra of 3rdgeneration G3 dendrimers. The spectrums comprise generally similar and expected characteristic absorption bands. The evaluation is given by indicating the bands in the FA-G3 FTIR spectra, and since the absorption bands of the NH bond stretching vibrations of the FA molecule are found in the 3500-3100 cm-1region in the spectra, interfering with the secondary amine peaks in the dendrimer structure, this region is seen with a peak at 3422 cm-1. The absorption band belonging to the phenyl and pterine ring vibrations in the molecular structure of FA cannot be seen in the FAGS FTIR spectra as it is located within the unseparated long band with two peaks at 1645 and 1618 cm-1. Sharp long band that causes absorption at -1693 cm'1, which belongs to the stretching vibrations of the C=O bonds of the COOH groups in the FA molecule and The intense band resulting from the structural asymmetric stretching vibrations of COOH groups causing absorption at ~1485 cm-1are not seen as expected in the FA-G3 FTIR spectra due to the reaction of these groups in the formation of conjugates. As a result, FTIR spectra confirm that FA-G3 synthesis was successful.

[0104] The structure of the 2ndgeneration G2 and 3rdgeneration G3 dendrimers is examined in detail by1H NMR analysis. The data obtained according to1H NMR results confirm the FTIR spectra results and show that the 2ndgeneration G2 and 3rdgeneration G3 dendrimers were successfully synthesised.1H NMR results of G2 dendrimer comprises 5 s, 8.20 (NHC(NH2)NH- NH2C(NH)NH-), dq, 7.94 (-NH(NH)CNHCH2-), dt, 8.17 (CCONHCH2-), ddt, 7.75 (-CCONHC(NH)NH-), ddt, 7.62 (-NH(NH2)CNH), m, 3.09 [(- NHCH2CH2-), (-COCH(NH2)CH2-], S, 3.62 (NH2C-), d, 2.93 (-CH2CH(NH2)CO-), m, 1 .46 (-CH2CH2CH2-)) (G3 igin 5 m, 8.18 [(NHC-), (NH2C(NH)NH-)] s, 8.08 [(-CONHCH- ), (-C(NH)NHCO-)], m, 7.95, [(-CH2NHC(NH)-), (-CONHC-)], ddd, 7.62 (- CH2NHC(NH)-), ddd, 7.75 (-CONHC(NH)-), s, 3.62 [(NH2C-), (-CH2NHC(NH)-)], m, 3.09 [(-CH2CH(NH2)CO-), (-CH2CH2NH-)] peaks.

[0105] REFERENCES

[0106] [1] Majoros, I., Jr, J.B., 2008, General Carriers for Drug Delivery, Dendrimer based nanomedicine, Chapter 2, Pan Stanford Publishing Pte. Ltd, Singapore, ISBN-13 978- 981-4241 -04-5.

[0107] [2] Esfand, R., Tomalia, D.A., 2001 , Poly(amidoamine) (PAMAM) dendrimers: From biomimicry to drug delivery and biomedical applications, Drug Discov Today, 6,427- 36.

[0108] [3] Namazi H. “Polymer in our Daily life”. BioImpacts, 2017, 7(2), 73-74

[0109] [4] Tabatabaei Mirakabad, F.S., Khoramgah, M.S., Keshavarz F., K., Tabarzad, M., Ranjbari, J. “Peptide dendrimers as valuable biomaterials in medical sciences”. Life Sci, 233,116754, 2019

[0110] [5] Sadler, K., Tam, J.P. “Peptide dendrimers: Applications and synthesis”. Rev Mol Biotechnol, 90,195-229, 2002

[0111] [6] Oktav Bulut M, Serdar SG, Koyuturk A, Cire A. “Dendrimerler ve Tekstilde Yeni Uygulamalan”. YEKARUM e-DERGi (Journal of YEKARUM), Yil 2016 / Cilt 3 / Sayi 2

[0112] [7] Saymer O, Qomoglu T. “NANOTA§IYICI SISTEMLERDE HEDEFLENDIRME” Ankara Ecz. Fak. Derg. / J. Fac. Pharm. Ankara, 40(3):62-79,2016

Claims

CLAIMS1. A nanoparticle system with cytotoxic properties against cancer cells, especially HeLa cancer cells, to be used in active targeting, comprising any one of- 1stgeneration G1 peptide dendrimer with the chemical structure ofFormula I, orFormula I- 2ndgeneration G2 peptide dendrimer with the chemical structure ofFormula II, orFormula II- 3rdgeneration G3 peptide dendrimer with the chemical structure ofFormula III, orFormula III4thgeneration G4 peptide dendrimer with the chemical structure ofFormula IV, orFormula IV3rdgeneration G3 peptide dendrimer modified with folic acid (FA) with the chemical structure of Formula V, the core of all of which consists of ethylene diamine (EDA), and the branching units and surface functional groups of all of which consist only of L-arginineFormula V- and [3-ionone (4-(2,6,6-Trimethyl-1 -cyclohexenyl)-3-buten-2-one).

2. A nanoparticle system with cytotoxic properties against cancer cells, especially HeLa cancer cells, to be used in active targeting according to Claim 1 , wherein1H NMR results of the 2ndgeneration G2 dendrimer comprise 5 s, 8.20 (NHC(NH2)NH-, NH2C(NH)NH-), dq, 7.94 (- NH(NH)CNHCH2-), dt, 8.17 (CCONHCH2-), ddt, 7.75 (-CCONHC(NH)NH-), ddt, 7.62 (-NH(NH2)CNH), m, 3.09 [(-NHCH2CH2-), (-COCH(NH2)CH2-], s, 3.62 (NH2C-), d, 2.93 (-CH2CH(NH2)CO-), m, 1.46 (-CH2CH2CH2-)) peaks and1H NMR results of the 3rdgeneration G3 dendrimer comprise (5 m, 8.18 [(NHC-), (NH2C(NH)NH-)] S, 8.08 [(-CONHCH-), (-C(NH)NHCO-)], m, 7.95, [(-CH2NHC(NH)-), (-CONHC-)], ddd, 7.62 (-CH2NHC(NH)-), ddd, 7.75 (- CONHC(NH)-), s, 3.62 [(NH2C-), (-CH2NHC(NH)-)], m, 3.09 [(- CH2CH(NH2)CO-), (-CH2CH2NH-)] peaks.

3. A nanoparticle system according to claim 1 or claim 2 for use in the treatment of cervical cancer.

4. The synthesis method of a nanoparticle system with cytotoxic properties against cancer cells, especially HeLa cancer cells, to be used in active targeting, comprising the process steps of: i. Protecting the amine groups of the L-Arginine molecule with N-Boc, ii. Synthesising the 1thGeneration G1 * dendrimer with the chemical structure of Formula VI,Formula VI iii. Obtaining the 1stgeneration G1 with the chemical structure of Formula I by removing the N-Boc protection in the 1stgeneration G1 * dendrimer,Formula I iv. Synthesising the 2ndGeneration G2* dendrimer with the chemical structure of Formula VII,Formula VII v. Obtaining the 2ndgeneration G2 with the Formula II chemical structure by removing the N-Boc protection in the 2ndgeneration G2* dendrimer,Formula II vi. Synthesising the 3rdGeneration G3* dendrimer with the chemical structure of Formula VIII,T1Formula VIII vii. Obtaining the 3rdgeneration G3 with the Formula III chemical structure by removing the N-Boc protection in the 3thgeneration G3* dendrimer,Formula III viii. Synthesising the 4ndGeneration G4* dendrimer with the chemical structure of Formula IX,Formula IX ix. Obtaining the 4rdgeneration G4 with the Formula IV chemical structure by removing the N-Boc protection in the 4thgeneration G4* dendrimer,Formula IV x. Obtaining the G3-FA complex with the chemical structure of FormulaV by modification of the 3rdgeneration G3 dendrimer with folic acid,Formula V xi. Formation of nanoconjugate structure by drug loading into dendrimers.

5. A method according to claim 4, wherein formation of nano conjugate structure by drug loading to the mentioned dendrimers in the process step (xi) comprisesAdding nonionic surfactant (Tween-80) with a G:Tween-80 ratio of 1 :1.2 (w / w) to the mixer and mixing for 1 hour while the product dissolved in water was mixed at 400 rpm in a magnetic stirrer so that the concentration of 2ndgeneration G2 and 3rdgeneration G3 peptide dendrimers in the final particle solution was 1 mg / mL,After mixing, adding the solution dissolved in 200 pL ethanol and containing Bl at a ratio of 1 :0.9 or 1 :1 or 1 :1.1 or 1 :1 .2 or 1 :1 .3 or 1 :1 .5 to G:BI by mass dropwise, andMeasuring nanoparticle sizes after mixing for 1 hour or 2 hours or 3 hours.

6. A method according to claim 5, wherein formation of nano conjugate structure by drug loading to the mentioned dendrimers in the process step (xi) comprisesAdding nonionic surfactant (Tween-80) with a G:Tween-80 ratio of 1 :1.2 (w / w) to the mixer and mixing for 1 hour while the product dissolved in water was mixed at 400 rpm in a magnetic stirrer so that the concentration of 2ndgeneration G2 and 3rdgeneration G3 peptide dendrimers in the final particle solution was 1 mg / mL,After mixing, adding the solution dissolved in 200 pL ethanol and containing Bl at a ratio of 1 :1 to G:BI by mass dropwise, and Measuring nanoparticle sizes after mixing for 2 hours.

Images